Background <p>Betulinic acid is a naturally occurring pentacyclic triterpenoid that has significant therapeutic potential, especially in the treatment of cancer, viral infections, and inflammatory diseases. However, poor water solubility and bioavailability are its major drawbacks for clinical applications. To overcome these problems, microspheres of betulinic acid were developed as a controlled-release drug delivery system.</p> Objectives <p>The present study aimed to develop and evaluate betulinic acid-loaded microspheres as a targeted drug delivery system for the treatment of inflammatory ulcerative colitis.</p> Methodology <p>The microspheres were prepared using ethyl cellulose and Eudragit RS-100, a pH-dependent methacrylic polymer that dissolves at pH &gt; 7, to achieve colon-specific delivery of betulinic acid, optimised by Box-Behnken Design (BBD) response surface methodology to determine particle size and entrapment efficiency. Characterisation of microspheres through FTIR, FESEM, and DSC evidenced that betulinic acid was successfully incorporated into the microspheres, exhibiting stability and amorphous nature within the polymer matrix. </p> Results <p>In vitro drug release studies indicated 97.4% release of the drug, betulinic acid, in 24&#xa0;h. Treatment with betulinic acid-loaded microspheres markedly attenuated both body weight loss (2.66 ± 0.51%) and colonic lesion scores (2.83 ± 0.40) when compared with the diseased control group. This treatment also significantly improved stool consistency to 2.16 ± 0.40, further emphasising the potential of the formulation as a treatment. Significant improvement in the colonic inflammation and lesion scores was noted for the microspheres as evaluated using an ulcerative colitis rat model in comparison with the control. </p> Conclusion <p>It can be concluded that betulinic acid-loaded microspheres providing a stable and efficient formulation that improves the bioavailability and therapeutic activity of the compound.</p>